Alzheimer's disease (AD) is a degenerative brain disorder that is characterized clinically by progressive loss of memory and cognitive impairment. Pathologically, the disease is characterized by lesions comprising neurofibrillary tangles, cerebrovascular amyloid deposits, and neuritic plaques. The cerebrovascular amyloid deposits and neuritic plaques contain amyloid-β peptide. The aggregation of amyloid-β peptide may be instrumental in the pathogenesis of AD.
Amyloid-β peptide is derived from amyloid-β precursor protein (APP). APP is a ubiquitous type 1 membrane protein (Kang et al., 1987, Nature 325:733-736; Kitaguchi et al., 1988, Nature 331:530-532; Tanzi et al., 1988, Nature 331:528-30) that is physiologically processed by proteolytic cleavage. (Selkoe, 1998, Trends Cell Biol 8: 447-453; Bayer et al., 1999, Mol Psychiatry 4:524-528; Haass and De Strooper, 1999, Science 286:916-919; Wolfe and Haass, 2001, J Biol Chem 276:5413-5416). First, cleavage by α- or β-secretases releases the large extracellular portion of APP. Subsequently, the remaining sequences of APP composed of a small extracellular stub, the transmembrane region (TMR), and the cytoplasmic tail are digested by γ-secretase at multiple positions. See Sastre et al., 2001, EMBO Rep 2:835-841; Yu et al., 2001, J Biol Chem 276:43756-43760. γ-Cleavage liberates an intracellular cytoplasmic fragment that may be translocated to the nucleus (Cupers et al., 2001, J Neurochem 78:1168-1178; Kimberly et al., 2001, J Biol Chem 276:40288-40292) and may function as a transcriptional activator (Cao and Südhof, 2001, Science 293:115-120; Gao and Pimplikar, 2001, Proc Natl Acad Sci USA 98:14979-14984). In addition, γ-cleavage generates small peptides derived from the TMR and adjacent extracellular sequences that include Aβ40 and Aβ42 which form the amyloid fibrils in Alzheimer's disease. See Glenner and Wong, 1984, Biochem Biophys Res Commun 122:1131-1135; Masters et al., 1985, EMBO J 4:2757-2763; reviewed in Selkoe, 1998, Trends Cell Biol 8: 447-453; Haass and De Strooper, 1999, Science 286:916-919.
The γ-cleavage of APP is mediated by presenilins, intrinsic membrane proteins that may correspond to γ-secretase and that are mutated in some cases of familial AD. See, e.g., Esler et al., 2000, Nat Cell Biol 2:428-434. Also, γ-cleavage occurs in APP homologs that are not implicated in AD. For example, Notch proteins are membrane proteins that are also cleaved in the middle of the TMR in a presenilin-dependent reaction. See, e.g., Yea et al., 1999, Nature 398:525-529; De Strooper et al., 1999, Nature 398:518-522; Struhl et al., 1999, Nature 398:522-525. Notch proteins are cell-surface proteins involved in intercellular signaling in which presenilin-dependent cleavage liberates a cytoplasmic fragment that functions in nuclear transcription. Struhl et al., 2000, Mol Cell 6:625-636. Sterol regulatory element binding proteins (SREPPs) are also cleaved to generate nuclear transcription factors. Brown et al., 2000, Cell 100:391-398. These observations suggested that the short cytoplasmic tail fragment of APP also may function as a transcriptional activator, and that feedback loops may exist between the nucleus and the cytoplasm or cell membrane whereby the rate of APP proteolysis is regulated.
This first of these hypotheses was confirmed by the findings that the short cytoplasmic tail of APP contains an NPTY (SEQ ID NO: 17) sequence that binds to phosphotyrosine binding (PTB) domains in multiple proteins, including Fe65 and Mints/X11a. See Fiore et al., 1995, J Biol Chem 270:30853-30856; Borg et al., 1996, Mol Cell Biol 16:6229-6241; Guenette et al., 1996, Proc Natl Acad Sci USA 93:10832-10837; McLoughlin and Miller, 1996, FEBS Lett 397:197-200; Zhang et al., 1997, EMBO J. 16: 6141-6150. Fe65 is an adaptor protein that forms a transcriptionally active complex with the released APP tail and a nuclear histone acetyltransferase, Tip60. See Cao and Südhof, 2001, Science 293:115-120.
In published United States patent application 20010034884, Peraus modified APP to create an APP fusion protein that incorporated a Gal4 binding domain and a VP16 transactivating domain, so that the rate of formation of the cytoplasmic tail of APP could be monitored by measuring the level of expression of a reporter gene whose transcription was controlled by a regulatable promoter containing Gal 4 binding sites. However, in contrast to the study of Cao and Südhof (2001), Peraus expressed no appreciation that the cytoplasmic tail of APP, without modification, acted as a transcription factor under physiological conditions, through an interaction with Fe65 and Tip60.
As introduced above, the NPTY sequence in the cytoplasmic tail fragment of APP can facilitate the binding of this protein to the PTB domains that are present in proteins of the Mints/X11 family. Mints 1 and 2 are genes initially identified as candidates for Friedreich's ataxia. Based on partial sequence analysis, these genes were thought to be orthologs. See Duclos and Koenig, 1995, Mamm Genome 6: 57-58. However, the sequencing of full-length cDNAs showed that the encoded proteins were products of distinct genes. See Okamoto and Südhof, 1997, J Biol Chem 272:31459-31464. To prevent confusion among different types of X11s, these proteins were named Mints 1 and 2. A third isoform was dubbed Mint 3. See Okamoto and Südhof, 1997, J Biol Chem 272:31459-31464; Okamoto and Südhof, 1998, Eur J Cell Biol 77:161-165. Subsequent recloning of the same proteins led to further renaming, and they are now also variably referred to as X11α/β/γ, mLin-10s, X11a/b/c, or X11L1/L2.
Mints/X11 proteins are composed of a long isoform-specific N-terminal sequence, a central PTB domain, and two C-terminal PSD-95, Drosophila disc large, zona occludens (PDZ) domains. Mint proteins interact with several other proteins in addition to APP. Mint 1 (but not Mints 2 or 3) binds to calcium/calmodulin-dependent serine protein kinase (CASK) (Butz et al., 1998, Cell 94:773-782), another adaptor protein (Hata et al., 1996, J Neurosci 16: 2488-2494). In C. elegans, CASK and Mint 1 homologs are encoded by the Lin-2 and Lin-10 genes whose mutation causes similar vulvaless phenotypes, suggesting that the Mint 1/CASK complex is evolutionarily conserved. See Butz et al., 1998, Cell 94:773-782; Kaech et al., 1998, Cell 94:761-771; Borg et al., 1998b, J Biol Chem 273:31633-31636; and Borg et al., 1999, J Neurosci 19:1307-1316. Mints 1 and 2 also bind to Munc18-1, an essential fusion protein at the synapse (Okamoto and Südhof, 1997, J Biol Chem 272:31459-31464; Biederer and Südhof, 2000, J Biol Chem. 275:39803-39806; Verhage et al., 2000, Science 287:864-869), and to presenilins which are intrinsic components of the γ-secretase (Lau et al., 2000, Mol Cell Neurosci 16:557-565).
The functions of the Mint proteins remain obscure. In C. elegans, Lin-10 (Mint 1) mediates the correct targeting of EGF-like receptors to the basolateral membrane of vulval precursor cells (Whitfield et al., 1999, Mol Biol Cell 10:2087-2100), and is necessary for delivery of AMPA-like glutamate receptors to synapses (Rongo et al., 1998, Cell 94:751-759). These data suggest that Lin-10/Mint 1 functions in membrane traffic of proteins to specific plasma membrane domains. In vertebrates, however, a variety of somewhat contradictory functions for Mints have been proposed. Transfection experiments revealed that Mints alter production of Aβ peptides, indicating a role in APP cleavage. See Borg et al., 1998a, J Biol Chem 273:14761-14766; Sastre et al., 1998, J Biol Chem 273:22351-22357; Mueller et al., 2000, J Biol Chem 275:39302-39306. In contrast, an interaction of Mint 1 with KIF17 in vitro led to the proposal that Mint 1 functions in trafficking neuronal NMDA-, but not AMPA-type glutamate receptors in vertebrates. See Setou et al., 2000, Science 288:1796-1802. This study renamed Mint 1 “mLin-10” in analogy to the C. elegans gene, but did not reference the previous finding that in nematodes Lin-10 only affects AMPA- but not NMDA-receptors (Rongo et al., 1998, Cell 94:751-759).
The small size of the APP cytodomain and the overlapping of its regions involved in the binding of Fe65 and the Mints proteins suggest that the latter may be involved in the competitive regulation of the intracellular signaling events mediated by the cytoplasmic tail of APP. Because it is likely that the rate of APP proteolysis is directly or indirectly regulated through feedback mechanisms that operate between the nucleus and the cytoplasm or cell membrane, a better understanding of the interactions between the cytoplasmic tail of APP and the Mints proteins will expand our knowledge of the mechanisms whereby the rate of APP proteolysis is regulated, thereby leading to greater insight into the pathophysiology of AD.
These studies may also facilitate the development of new methods of treatment for this disease. At present, the only medications approved by the U.S. Food and Drug Administration for the treatment of AD are cholinesterase inhibitors. Unfortunately, these drugs provide only limited clinical benefit in controlling the symptoms of AD, and do little to actually intervene in the disease process. Thus, there is a strong need for the development of better treatments for AD.
In accordance with the present invention, it has been discovered that, while all three Mint proteins (Mints 1-3) bind to the cytoplasmic tail of APP, only Mint 1 and 2 can modulate the transcriptional activation mediated by a fusion protein consisting of the cytoplasmic tail of APP coupled to the transcription factor Gal4/VP16. These findings suggest that Mints 1 and 2, variants of these proteins that display enhanced abilities to modulate the transcriptional activation mediated by the cytoplasmic tail of APP relative to wild-type Mints 1 and 2, or small molecules which either mimic, inhibit or potentiate the effects of these proteins in modulating the transcriptional activation mediated by the cytoplasmic tail of APP, may be useful in regulating the rate of APP proteolysis and hence in the treatment of AD.